Multidrug resistance in bacteria has become more the norm than the unusual. While characteristically mediated by plasmids and transposons, increasing evidence shows that chromosomal (intrinsic) regulatory genes and muitidrug effiux pumps mediate resistance to multiple antibiotics and hazardous substances. One such regulatory system is the marRAB operon discovered in Escherichia coil whose MarA protein product controls expression of over 80 genes in the mar regulon. Initially described as an activator, MarA appears to have direct repressor activity as well. Homologs of the E.cofi MarA and MarR (the repressor of the mar operon) have been found in many different genera including both gram positive and gram negative organisms. Studies of Salmonella and E.cofi reveal not only mar-mediation of drug resistance, but also of colonization and virulence. The DNA binding sites for MarR and MarA have been identified as well as their crystal structures. Still the molecular and biochemical elements which define MarA activation or repression of different genes is not understood, nor is the regulation of the operon by genes other than MarR. To improve knowledge about the molecular control and activity of the mar regulon in E.cofi and other clinically important bacteria, this proposal seeks to: 1) determine the molecular basis for the difference between negative and positive transcriptional control by the MarA protein of genes in the mar regulon; studies in vitro and in vivo will define the sequence, orientation and location of the regulatory DNA sequences near the promoters of particular genes in the regulon. 2) enhance understanding of MarR function through two-hybrid studies of its interaction with other proteins, use of macro/micro arrays to determine possible regulation of other loci by MarR, and determination of the crystal structure of MarR with DNA or a ligand. 3) identify other chromosomal genes besides marR which regulate expression of the mar operon. 4) investigate mar loci in other bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Yersinia pestis. Studies of the E.cofi marRAB serve as a paradigm for insights into related genetic consequence to human health. Improved understanding will help to suggest novel and curing infections.